[目的]探讨miR-935靶向GLUD1调控LKB1缺失型肺癌细胞失巢凋亡的潜在机制。[方法]分离条件下培养肺癌细胞构建失巢细胞模型，抽取各个分离时间点的细胞总RNA进行miRNA-seq，过表达或敲低差异miRNA后检测LKB1缺失型肺癌细胞A549和H157的失巢凋亡水平。通过miRDB在线分析和遗传学筛选鉴定miRNA的关键底物。根据是否过表达miR-935分为对照组和miR-935过表达组;根据是否敲低GLUD1分为对照组和GLUD1敲低组。[结果]随着分离时间的延长，肺癌细胞中miR-935的表达水平下降(1。47±0。15 vs0。09±0。01，P＜0。05)、GLUD1的表达水平上升(0。87±0。16 vs 1。44±0。21，P＜0。05)。过表达miR-935后，肺癌细胞的失巢凋亡水平上升[(15。87± 2。23)％vs(49。79±7。63)％，P＜0。05]。敲低GLUD1后，肺癌细胞的失巢凋亡水平显著上升[(16。32±3。11)％vs(48。21±5。67)％，P＜0。05]。过表达miR-935后，肺癌细胞中GLUD1 mRNA和蛋白的表达水平下降(0。87±0。16 vs 0。10±0。01，P＜0。05)。miR-935与GLUD1 mRNA的3'UTR有相互作用。敲低GLUD1后，肺癌细胞内α-酮戊二酸(α-KG)的水平显著下降[(59。32±6。13)μmol/L vs(41。22±3。93)μmol/L，P＜0。05]。敲低 GLUD1 后加入 methyl-α-KG，A549和H157细胞的失巢凋亡水平回到了与未敲低GLUD1细胞的相似水平。[结论]miR-935靶向GLUD1 mRNA的3'UTR并减少了 GLUD1的表达水平，促进了 LKB1缺失型肺癌细胞的失巢凋亡。
Mechanism of miR-935 targeting GLUD1 to regulate anoikis in LKB1-deficient lung cancer cells
[Objective]To investigate the potential mechanism of miR-935 targeting GLUD1 to regulate anoikis in LKB1-deficient lung cancer cells.[Method]Lung cancer cells were cultured under isolated conditions to construct a lost nest cell model.miRNA-seq was performed by extracting total RNA from cells at each isolation time point,and the levels of lost nest anoikis in LKB1-deficient lung cancer cells A549 and H157 were detected after overexpression or knockdown of differential miRNA.Key substrates of miRNAs were identified by miRDB online analysis and genetic screening.They were divided into con-trol group and miR-935 overexpression group according to whether miR-935 was overexpressed or not.They were divided in-to control group and GLUD1 knockdown group according to whether GLUD1 knockdown or not.[Result]With the extension of isolation time,the expression level of miR-935 decreased(1.47±0.15 vs 0.09±0.01,P＜0.05),and the expression level of GLUD1 increased(0.87±0.16 vs 1.44±0.21,P＜0.05).After overexpression of miR-935,the level of anoikis of lung canc-er cells increased[(15.87±2.23)％vs(49.79±7.63)％,P＜0.05].After GLUD1 knockdown,the apoptosis level of lung cancer cells was significantly increased[(16.32±3.11)％vs(48.21±5.67)％,P＜0.05].After overexpression of miR-935,GLUD1 mRNA and protein expression levels in lung cancer cells were decreased(0.87±0.16 vs 0.10±0.01,P＜0.05).miR-935 interacts with the 3'UTR of GLUD1 mRNA.After GLUD1 knockdown,the intracellular α-ketoglutarate(α-KG)level of lung cancer was significantly decreased[(59.32±6.13)μmol/L vs(41.22±3.93)μmol/L,P＜0.05].With the addition of methyl-α-KG after GLUD1 knockdown,the loss of nests apoptosis levels in A549 and H157 cells re-turned to similar levels to those in non-knockdown GLUD1 cells.[Conclusion]miR-935 targeted the 3'UTR of GLUD1 mR-NA and reduced the expression level of GLUD1,promoting anoikis in LKB1-deficient lung cancer cells.
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